Cryo-STEM Tomography of Intact Vitrified Fibroblasts

Kirchenbüechler, David; Mutsafi, Yael; Horowitz, Ben; Levin-Zaidman, Smadar; Fass, Deborah; Wolf, Sharon G.; Elbaum, Michael

doi:10.3934/biophy.2015.3.259

Cited by 18 publications

(16 citation statements)

References 28 publications

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“…The approach is now established for application to other metal-binding macromolecules such as enzymes, ion channels, and nucleic acids. Faithful identification of heavy atoms will also be essential in detection of metal tags as protein labels for future cryo-microscopy and tomography in the cellular context (19,20,39,40).…”

Section: Discussionmentioning

confidence: 99%

“…However, low contrast and high electron dose have hindered its application to imaging of fully hydrated, vitrified protein or cellular samples. This hurdle has recently been surmounted by careful setting of the illumination and collection angles and implementation of low-dose protocols (18)(19)(20). Cryo-STEM tomography (CSTET) provides distinct advantages for the study of thick specimens.…”

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confidence: 99%

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Detection of isolated protein-bound metal ions by single-particle cryo-STEM

Elad

Bellapadrona

Houben

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Detection of isolated protein-bound metal ions by single-particle cryo-STEM

Elad

Bellapadrona

Houben

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Tomography by scanning TEM (STEM) was introduced for plastic embedded section (16) and offers an alternative to widefield phase contrast for cryotomography (17)(18)(19)(20). In STEM, a focused probe is scanned across the specimen while independent detectors record the scattered (dark field) and unscattered (bright field) signals.…”

Section: Significancementioning

confidence: 99%

Three-dimensional deconvolution processing for STEM cryotomography

Waugh

Wolf

Fass

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.

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“…Tomography by scanning TEM (STEM) was introduced for plastic embedded section (16) and offers an alternative to wide field phase contrast for cryo-tomography (17)(18)(19)(20). In STEM, a focused probe is scanned across the specimen while independent detectors record the scattered (dark-field) and unscattered (bright-field) signals.…”

Section: Introductionmentioning

confidence: 99%

3D Deconvolution Processing for STEM Cryo-Tomography

Waugh

Wolf

Fass

et al. 2020

Preprint

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The complex environment of biological cells and tissues has motivated development of three dimensional imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryo-scanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles, and then in representative cryo-tomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.Significance statementElectron tomography is used to reveal the structure of cells in three dimensions. The combination with cryogenic fixation provides a snapshot in time of the living state. However, cryo-tomography normally requires very thin specimens due to image formation by conventional phase contrast transmission electron microscopy (TEM). The thickness constraint can be relaxed considerably by scanning TEM (STEM), yet three-dimensional (3D) reconstruction is still subject to artifacts inherent in the collection of data by tilted projections. We show here that deconvolution algorithms developed for fluorescence microscopy can suppress these artifacts, resulting in significant contrast enhancement. The method is demonstrated by cellular tomography of complex membrane structures, and by segmentation of chromatin into distinct, contiguous domains of heterochromatin and euchromatin at high and low density, respectively.

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Cryo-STEM Tomography of Intact Vitrified Fibroblasts

Cited by 18 publications

References 28 publications

Detection of isolated protein-bound metal ions by single-particle cryo-STEM

Detection of isolated protein-bound metal ions by single-particle cryo-STEM

Three-dimensional deconvolution processing for STEM cryotomography

3D Deconvolution Processing for STEM Cryo-Tomography

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